Although the most common genetic cause of congenital hearing impairment is mutation in GJB2, a significant portion of the mutation load has not yet been discovered. Diagnostic and research labs have observed an excess of individuals with hearing loss who are identified with a GJB2 mutation on only one chromosome, for whom, consequently, a definitive diagnosis of genetic etiology cannot be made. Our lab identified and characterized a DFNB1 allele that contains a 131-kb deletion with a proximal breakpoint well away from the transcriptional start sites of both GJB2 and GJB6; this deletion segregates with reduced or lost expression of both GJB2 and GJB6 mRNA. The evidence of this and other important DFNB1 deletion alleles suggests that cis-regulatory elements for GJB2 and GJB6 exist at considerable distance from the genes themselves. We hypothesize that single nucleotide variants and small deletions affecting the function of distant enhancer elements constitute the 'missing' mutation among individuals with hearing loss who are heterozygous for a single DFNB1 mutation. We propose to advance the identification of missing DFNB1 mutation by a two-pronged approach. First, we propose to target next-generation sequencing to the DFNB1 locus, including the 131-kb interval in which GJB2-regulatory function has been documented. We will compare the sequences of 95 people with hearing loss and monoallelic mutation of GJB2 to three other sequence data sets: 96 population-matched controls, 1 hearing person in whom we have documented expression of both GJB2 and GJB6 mRNA, and any/all publicly available genomes (e.g. 1000 Genomes Project). These comparisons will yield a set of candidate regulatory mutations from the DFNB1 locus, that we hypothesize will comprise either one or several variants that are statistically over-represented among our patients, or a collection of novel variants that map as one or more clusters. Second, by chromosome conformation capture carbon-copy (5C) in a number of proxy cell lines, we will identify regions from within the DFNB1 locus that interact specifically with DNA at or near the GJB2 promoter. We will intersect the findings from each of these aims with consideration of public datasets of bioinformatically-predicted and experimentally-determined regulatory function to prioritize candidate regulatory regions, and associated candidate variants, for functional analysis by reporter assay. Interrogation of the DFNB1 locus by these several strategies will reveal previously unidentified DFNB1 mutation.